Device and method for observing a fluorescent sample

Abstract

A method for observing a fluorescent sample, the sample comprising a fluorescent agent that emits fluorescence light, in a fluorescence spectral band, when it is illuminated by excitation light, in an excitation spectral band, the method comprising the following steps: a) placing the sample on a holder; b) illuminating the sample, with an excitation light source, in the excitation spectral band, the light emitted by the light source propagating along a propagation axis; c) detecting fluorescence light, in the fluorescence spectral band, with an image sensor; the method being such that the holder comprises a thin layer formed from a first material, of a first refractive index, the thin layer lying in a holder plane perpendicular to the propagation axis, the thin layer comprising a first photonic crystal and second photonic crystals configured to confine the excitation light and the fluorescence light in the thin layer.

The invention claimed is: 
     
       1. A method for observing a fluorescent sample, the sample comprising a fluorescent agent that emits fluorescence light, in a fluorescence spectral band, when it is illuminated by excitation light, in an excitation spectral band, the method comprising:
 a) placing the sample against a holder; 
 b) illuminating the sample, with a light source, in the excitation spectral band, the light emitted by the light source propagating along a propagation axis; 
 c) detecting fluorescence light, in the fluorescence spectral band, with an image sensor; 
 wherein 
 the holder comprises a thin layer formed from a first material, of a first refractive index, the thin layer lying in a holder plane perpendicular to the propagation axis, 
 the holder plane extending along a longitudinal axis, the thin layer comprising:
 first inclusions formed from a second material, of a second refractive index different from the first refractive index, the first inclusions defining a first periodic pattern, the first inclusions being distributed with a first period, parallel to the longitudinal axis, the first inclusions forming a first photonic crystal that is resonant in the excitation spectral band; 
 second inclusions formed from a third material, of a third refractive index different from the first refractive index, and forming a second periodic pattern, the second inclusions being distributed with a second period, parallel to the longitudinal axis, the second period being strictly longer than the first period, the second inclusions forming a second photonic crystal that is resonant in the fluorescence spectral band; 
 at least two second photonic crystals being placed on either side of a first photonic crystal; 
 
 such that:
 part of the light emitted by the light source, at the excitation wavelength, is confined in the thin layer, by the first photonic crystal, resulting in a formation and an amplification of a light wave that propagates at an interface between the thin layer and the sample, at the excitation wavelength; 
 the fluorescence light propagates in the thin layer, through a first photonic crystal, while being reflected by the second photonic crystals placed on either side of the first photonic crystal, resulting in an amplification of the fluorescence light. 
 
 
     
     
       2. The method of  claim 1 , wherein the sample is placed in contact with the thin layer, or at a distance from the latter smaller than 1 mm. 
     
     
       3. The method of  claim 1 , wherein
 the first photonic crystal is one-dimensional, the first inclusions extending parallel to a lateral axis perpendicular to the longitudinal axis; 
 and/or the second photonic crystal is one-dimensional, the second inclusions extending parallel to the lateral axis. 
 
     
     
       4. The method of  claim 1 , wherein
 the first photonic crystal is two-dimensional, the first inclusions being spaced apart from one another, along a lateral axis perpendicular to the longitudinal axis; 
 and/or the second photonic crystal is two-dimensional, the second inclusions also being spaced apart from one another; along the lateral axis. 
 
     
     
       5. The method of  claim 1 , wherein:
 the first period, parallel to the longitudinal axis; is comprised between 100 nm and 300 nm; 
 and/or the second period, parallel to the longitudinal axis, is comprised between 200 nm and 400 nm. 
 
     
     
       6. The method of  claim 1 , wherein the thickness of the thin layer, along the propagation axis, is comprised between 100 nm and 300 nm. 
     
     
       7. The method of  claim 1 , wherein the holder is placed between the sample and the image sensor. 
     
     
       8. The method of  claim 7 , wherein no image-forming optics are placed between the image sensor and the holder. 
     
     
       9. The method of  claim 1 , wherein the third material, forming the second inclusions, is identical to the second material, forming the first inclusions. 
     
     
       10. A device for observing a fluorescent sample; the sample comprising a fluorescent agent that emits fluorescence light, in a fluorescence spectral band, when it is illuminated by excitation light, in an excitation spectral band, the device comprising:
 a light source, configured to emit an illuminating light wave in the excitation spectral band, the light propagating; toward the sample, along a propagation axis; 
 a sample holder; configured to receive the sample; 
 an image sensor; configured to detect fluorescence light, emitted by the sample; in the fluorescence spectral band; 
 wherein: 
 the holder comprises a thin layer formed from a first material, of a first refractive index, the thin layer lying in a holder plane perpendicular to the propagation axis, 
 the holder plane extending along a longitudinal axis, the thin layer comprising:
 first inclusions formed from a second material, of a second refractive index different from the first refractive index, the first inclusions defining a first periodic pattern, the first inclusions being distributed with a first period, parallel to the longitudinal axis, the first inclusions forming a first photonic crystal that is resonant in the excitation spectral band, the first photonic crystal being configured to confine a portion of the illumination light wave in the thin layer; 
 second inclusions formed from a third material, of a third refractive index different from the first refractive index; and forming a second periodic pattern, the second inclusions being distributed with a second period, parallel to the longitudinal axis, the second period being strictly longer than the first period, the second inclusions forming a second photonic crystal that is resonant in the fluorescence spectral band; 
 at least two second photonic crystals being placed on either side of a first photonic crystal. 
 
 
     
     
       11. The device of  claim 10 , wherein:
 the first photonic crystal is one-dimensional, the first inclusions extending parallel to a lateral axis perpendicular to the longitudinal axis; 
 and/or the second photonic crystal is one-dimensional, the second inclusions extending parallel to the lateral axis. 
 
     
     
       12. The device of  claim 10 , wherein:
 the first photonic crystal is two-dimensional, the first inclusions being spaced apart from one another, along a lateral axis perpendicular to the longitudinal axis; 
 and/or the second photonic crystal is two-dimensional, the second inclusions being spaced apart from one another, along the lateral axis. 
 
     
     
       13. The device of  claim 10 , wherein:
 the first period, parallel to the longitudinal axis, is comprised between 100 nm and 300 nm; 
 and/or the second period is comprised between 200 nm and 400 nm. 
 
     
     
       14. The device of  claim 10 , wherein the thickness of the thin layer, along the propagation axis, is comprised between 100 nm and 300 nm. 
     
     
       15. The device of  claim 10 , wherein the holder is bounded by a top face, adjacent to the thin layer, and a bottom face, opposite to the top face, the image sensor being placed, facing the holder, closer to the bottom face than to the top face. 
     
     
       16. The device of  claim 10 , wherein no image-forming optics lie between the holder and the image sensor. 
     
     
       17. The device of  claim 16 , wherein the holder and the image sensor form the same component. 
     
     
       18. The device of  claim 10 , wherein the third material, forming the second inclusions, is identical to the second material, forming the first inclusions.